%0 Journal Article
%T A novel Gateway£¿-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens
%A Sy Traore
%A Bingyu Zhao
%J Plant Methods
%D 2011
%I BioMed Central
%R 10.1186/1746-4811-7-42
%X In this study, we constructed a novel Gateway£¿-compatible destination vector, pEG101-SacB/R, by replacing the ccdB gene with a SacB-SacR gene cassette as the negative selectable marker.Our results demonstrate that the new pEG101-SacB/R destination vector can be used for Gateway£¿ cloning in Agrobacterium tumefaciens. pEG101-SacB/R will be a valuable tool for high-throughput functional analysis of genes in planta.The wide-spread availability of genomic sequences from many organisms has raised interest in characterizing the biological functions of newly discovered genes through various high-throughput methodologies [1]. In order to study gene function, a gene-of-interest needs to be cloned into different plasmid vectors. In plant biology research, the gene-of-interest is frequently cloned into binary vectors that can be used for Agrobacterium-mediated transformation [2].Gateway£¿ technology was developed as a convenient and fast gene cloning system (Invitrogen£¿, Carlsbad, CA). This method involves three key steps: (1) amplifying the targeted gene by PCR; (2) directly cloning the PCR product into a TopoEntr/D£¿ vector without digestion/ligation; and (3) subcloning the targeted gene from the entry vector into any destination vector using the Gateway£¿ LR cloning technique. The Gateway£¿ LR cloning reaction is mediated by the site-specific homologous DNA recombination properties of bacteriophage lambda [3]. This method is more convenient than other methods because it does not involve either DNA digestion nor ligation, two processes that can hinder the cloning process [4]. The Gateway£¿-compatible destination vector harbors a negative selectable marker, the ccdB gene, which produces a toxic protein that is lethal to most E. coli strains, including DH5¦Á [5,6]. During the Gateway£¿ LR reaction, the ccdB gene in the destination vector is replaced by the targeted gene from an entry vector through site-specific homologous DNA recombination. The LR reaction mixture containing both rec
%U http://www.plantmethods.com/content/7/1/42